1. Field of Invention
The present invention concerns methods and compositions of use for the treatment of Alzheimer's disease (AD) in subjects, particularly in human subjects. In certain embodiments, the methods may comprise exposure of phage-displayed human antibodies, such as single-chain Fv (scFv) antibodies, to a human amyloid-beta (Aβ42) peptide and selection of anti-Aβ42 antibodies. In other embodiments, the compositions may comprise anti-Aβ42 antibodies, for example that have been prepared by the disclosed methods. In still other embodiments, the compositions may comprise one or more synthetic peptides, peptide mimetics and/or peptide analogs containing one or more amino acid sequences selected from one or more anti-Aβ42 antibodies. Methods of therapeutic treatment of a subject may comprise administration of anti-Aβ42 peptides, peptide analogs and/or peptide mimetics containing one or more amino acid sequences selected from one or more anti-Aβ42 antibodies to a subject with AD or a subject at risk of developing AD.
2. Description of Related Art
The accumulation of amyloid-beta (Aβ) peptide in the brain and its deposition as plaques has been hypothesized to play a central role in the neuropathology of Alzheimer's Disease (AD) (Selkoe, 2001; Thorsett and Latimer, 2000; Klein et al., 2001). Neurons in the brain produce Aβ fragments from a larger precursor molecule named amyloid precursor protein (APP). When released from the cell, Aβ fragments may accumulate in extracellular amyloid plaques. It appears that not only the well-known Aβ amyloid fibrils but much smaller soluble forms of aggregated Aβ fragments (protofibrils and small oligomers), that escape detection by methods suitable for fibrils, are involved in the pathogenesis of AD (Klein et al., 2001). This explains the poor correlation between fibrillar amyloid load and disease progression.
Aβ fragments are generated through the action of specific proteases within the cell. The most important among these enzymes are beta- and gamma-secretase. It has been proposed that small compounds that cross the blood-brain barrier (BBB) and decrease but do not eliminate either beta- or gamma-secretase activity may be of use for therapies in the early clinical phases of AD. But interfering with normal metabolic reactions of the organism, such as the action of beta- and gamma-secretase, is not desirable, and an alternative approach that targets a specific pathological event, Aβ deposition, would be more appropriate for effective treatment and prevention of AD.
There are previous reports of efforts to find compounds that selectively destroy Aβ plaques. Thus, it was reported that nitrophenols inhibited the aggregation of Aβ in vitro and caused disaggregation of previously formed amyloid fibrils (Feliche et al., 2001). Also, nitrophenols protected rat hippocampal neurons in culture from the neurotoxic effect of Aβ and inhibited the formation of Aβ deposits in rat hippocampi in an in vivo model system of cerebral amyloid deposits (Feliche et al., 2001). Fassbender et al. (2001) showed that the cholesterol-lowering drugs, simvastatin and lovastatin, reduce intracellular and extracellular levels of Aβ in primary cultures of hippocampal neurons and mixed cortical neurons, and that guinea pigs treated with high doses of simvastatin showed a strong and reversible reduction of cerebral Aβ. None of these compounds to date has provided an effective treatment or cure for human AD.
Alzheimer-type neuropathology has been observed in transgenic mice in which transgenes for human APP provided elevated brain levels of Aβ. Experiments using this mouse model of AD (PDAPP transgenic mouse model) have been used to investigate many questions related to Aβ and AD. Thus, it has been shown that immunization of PDAPP mice with Aβ peptide significantly reduced amyloid deposition and certain AD-like neuropathological features in old mice, and also essentially prevented amyloid formation, neuritic dystrophy and astrogliosis in young animals (Schenk et al., 1999; Janus et al., 2000; Morgan et al, 2000).
These results suggested that the immunization with Aβ may be effective in preventing and treating AD. Janus and collaborators showed that Aβ immunization reduced both deposition of cerebral fibrillar Aβ and cognitive dysfunction in the TgCRND8 murine model of AD (a mutant, K670N/M671L and V717F, human βAPP695 transgene expressed under the regulation of the Syrian hamster prion promoter on a C3H/B6 strain background) (Janus et al., 2000). Another study on Aβ immunization was performed by Morgan et al (2000). Those authors demonstrated that vaccination of transgenic mice with Aβ protected them from the learning and age-related memory deficits that normally occur in this mouse model of AD. The Aβ-vaccinated mice also exhibited a partial reduction in amyloid burden at the end of the study. These cumulative data suggest the use of Aβ immunization as a therapeutic approach that may prevent and, possibly, treat AD. However, in human clinical trials with Aβ42 immunization, some patients developed symptoms of brain inflammation and the phase 2A clinical trial was halted (Munch and Robinson, 2002). More recent results suggest that patients who developed significant antibody titers against Aβ42 did not demonstrate cognitive decline (Hock et al., 2003).
In the studies mentioned above a whole Aβ peptide incubated overnight in buffer was used for immunization of mice. Such Aβ solutions typically contain amyloid fibrils together with a mixture of smaller aggregates. Because of the low immunogenicity of the Aβ fibrils, repeated antigen administrations were required to obtain high levels of anti-Aβ antibodies. Moreover, immunizing with toxic fibrils may induce more accumulation of the toxic amyloid itself (Morgan et al., 2000).
Attempts to design other immunogens capable of inducing anti-Aβ antibodies with anti-aggregating properties were made. The first step in these studies was the identification of epitopes within the whole Aβ molecule to which anti-Aβ antibodies bind. Both classical synthetic peptide and phage display peptide library approaches have been applied. Thus, using a phage display peptide library it was shown that residues EFRH located at positions 3-6 of the N-terminal Aβ peptide comprised the epitope that was found to be the main regulatory site for fibril formation (Frenkel et al. 1998). Subsequently, filamentous phages displaying EFRH peptide were used as a specific and non-toxic immunogen in guinea pigs for the production of anti-aggregating antibodies (Frenkel et al., 2000). Those authors have shown that serum antibodies raised against EFRH phage prevented the Aβ neurotoxic effect and disaggregated Aβ fibrils.
One of the disadvantages of any active immunization procedure is the generation of very robust immune responses, particularly cellular immune response, that may not be desirable in an elderly patient population suffering from AD. The hypothesis that passive antibody immunotherapy could be more appropriate for these individuals promoted efforts towards the generation of antibody reagents that are capable of preventing and clearing amyloid aggregates. Monoclonal antibodies raised against Aβ fragments spanning amino acid residues 1-28, prevented the aggregation of Aβ and disaggregated Aβ fibrils in vitro (Solomon et al., 1996; Solomon et al. 1997). Splenocytes from actively immunized mice demonstrated a T-cell proliferative response to Aβ in vitro, indicating the possible involvement of T-cell immunity in the therapeutic effect of immunization.
It has been determined that peripherally administered polyclonal and monoclonal antibodies against Aβ as well as their Fab or scFv regions entered the central nervous system (CNS) and reduced plaque burden along with reduction of pathology in a mouse model for AD (Bard et al., 2000; Frenkel et al., 2000; Bacskai et al., 2002; Kotilinek et al., 2002). Those results indicate that in the absence of T-cell immunity, antibodies or their fragments are sufficient to decrease amyloid deposition and AD-like pathology via classical Fc-dependent phagocytosis or direct disruption of both soluble assemblies (Kotilinek et al., 2002) or fibrils of Aβ peptide. In addition, the reduction of brain Aβ burden by peripheral administration of the anti-Aβ monoclonal antibody m266, that was capable of facilitating the clearance of Aβ out of the CNS to plasma, has been reported (DeMattos et al., 2001).
However, in another study passive immunization also demonstrated adverse side effects, such as microhemorrhages, after passive administration of mouse monoclonal anti-amino terminal Aβ antibody in APP23 transgenic mice (Pfeifer et al., 2002). This mouse model exhibits the age-related development of amyloid plaques and neurodegeneration as well as cerebral amyloid angiopathy (CAA) similar to that observed in the human AD brain (Sturchler-Pierrat et al., 1997; Calhoun et al., 1999). A possible link may exist between adverse side effects noted in APP23 transgenic mice and neuroinflammatory complications of immunization seen in a human trial. Thus, a need exists for an AD therapy that would be effective to inhibit or reverse formation of Aβ fibrils in the brain, while exhibiting reduced side effects compared to presently available immunotherapies.